1. Field of the Invention
The present invention relates generally to the fields of molecular biology and gene cloning. More specifically, the present invention relates to the identification and characterization of the gene encoding human cytoplasmic polyadenylation element binding protein and uses thereof.
2. Description of the Related Art
During meiotic maturation of human oocytes gene tran-scription is repressed (Braude et al., 1988) and required proteins are translated from pre-existing, maternally derived mRNAs (Pal et al., 1994). In model systems (Drosophila, Xenopus, and the mouse), certain maternally derived mRNAs which encode key regulators of cell cycle progression and pattern formation are translationally silent in immature oocytes and become translationally activated following hormonal stimulation (Davidson, 1986; Wickens et al., 1996). This translational activation has been correlated with the cytoplasmic polyadenylation of the mRNAs, a process directed by two elements within the mRNA 3′ untranslated region (UTR) (reviewed in Richter, 1999). The first element is the AAUAAA polyadenylation hexanucleotide and the second element is a uridine-rich sequence of general consensus UUUUUAU termed the cytoplasmic polyadenylation element (CPE). In addition to directing cytoplasmic polyadenylation and translational activation, these cytoplasmic polyadenylation element sequences have also been implicated in mediating translational repression in immature oocytes (de Moor and Richter, 1999; Barkoff et al., 2000; Tay et al., 2000) and during the early phases of hormonally stimulated oocyte maturation (Charlesworth et al., 2000). Cytoplasmic polyadenylation element-mediated mRNA translational control has also been suggested to occur in mammalian neuronal cells (Wu et al., 1998).
A cytoplasmic polyadenylation element binding protein, CPEB, has been cloned from a number of species (Hake and Richter, 1994; Gebauer and Richter, 1996; Bally-Cuif et al., 1998; Walker et al., 1999) and has been implicated in mediating both polyadenylation-dependent translational activation and cytoplasmic polyadenylation element-directed translational repression (Hake and Richter, 1994; Stebbins-Boaz et al., 1996; Stutz et al., 1998; Minshall et al., 1999; Stebbins-Boaz et al., 1999; Mendez et al., 2000). While it is not clear how the cytoplasmic polyadenylation element binding protein can exert these apparently opposite effects on mRNA translation, there is some evidence that the C-terminal domain is necessary for translational repression while the N-terminal domain may regulate translational activation. It has been reported that overexpression of an N-terminally truncated form of the Xenopus cytoplasmic polyadenylation element binding protein (lacking the first 139 amino acids) did not significantly affect translational repression but did block both cytoplasmic polyadenylation and translational induction (Mendez et al., 2000).
Given the key role of cytoplasmic polyadenylation in the control of mRNA translation in model organisms, it is of interest to determine if a similar process occurred in humans. However, human cytoplasmic polyadenylation element binding protein has not been identified. Thus, the prior art is deficient in identifying a human cytoplasmic polyadenylation element binding protein which is essential for the study of mRNA translation control in human. The present invention fulfills this long-standing need and desire in the art by cloning a human cytoplasmic polyadenylation element binding protein.
Despite a critical role in the control of human fertility, the mechanisms regulating human oocyte maturation are not well characterized. In model organisms, accumulation of critical cell cycle regulatory proteins during oocyte meiotic maturation depends upon the regulated translation of maternally derived mRNAs (Wickens, et al., 2000; Mendez and Richter, 2001). The maternal mRNA encoding the Mos proto-oncogene is subject to tight translational regulation in oocytes from a variety of vertebrate species. The Mos protein is a serine/threonine kinase which activates the MAP kinase cascade through direct phosphorylation of the MAP kinase activator MEK (aka MAP kinase kinase) (Posada et al. 1993; Shibuya et al., 1996). In the mouse, Mos protein is absent from immature oocytes and maturation-dependent cytoplasmic polyadenylation correlates with the translational activation of the maternal Mos mRNA (Gebauer, et al., 1994). While mouse meiotic cell cycle progression does not depend on Mos translation, Mos protein function is necessary for arrest of the mature oocyte at meiotic metaphase II (Araki et al., 1996; Colledge et al. 1994; Hashimoto et al. 1994; Hashimoto, 1996). In addition to a requirement for meiotic metaphase II arrest (Sagata et al., 1989; Daar et al., 1991), Mos function is also required earlier during maturation of oocytes from the frog, Xenopus laevis, to mediate entry into Meiosis II after completion of Meiosis I in oocytes (Gross et al., 2000; Dupre et al., 2002).
Meiotic cell cycle progression has been best characterized in Xenopus, where the cytoplasmic polyadenylation and translational activation of select maternal mRNAs occur in a strict temporal order (Dupre et al., 2002; Hochegger et al., 2001; Howard et al., 1999; Nakajo et al., 2000; Sheets et al., 1994; Sheets et al., 1995; Ferby et al., 1999; Sagata et al., 1988; Charlesworth et al., 2000; Ballantyne et al., 1997; de Moor et al., 1997). The ability to regulate addition of a poly[A] tail extension in the oocyte cytoplasm requires a polyadenylation nucleotide sequence (typically AAUAAA) as well as additional 3′ UTR regulatory sequences, including cytoplasmic polyadenylation elements (CPE) (reviewed in Richter, 2000) and polyadenylation response elements (PRE) (Charlesworth et al., 2002; Charlesworth et al., 2004). CPE sequences have been shown to repress mRNA translation in immature oocytes and to direct temporally late cytoplasmic polyadenylation and translational activation in maturing oocytes. Both aspects of CPE function appear to require the CPE-binding protein (CPEB1) (Gebauer et al., 1994; Charlesworth et al., 2000; Fox et al., 1989; McGrew et al., 1989; McGrew et al., 1990; Paris and Richter, 1990; SallÈs et al., 1992; Standart and Dale, 1993; Stebbins-Boaz et al., 1996; Stutz et al., 1998; de Moor and Richter, 1999; Minshall et al., 1999; Barkoff et al., 2000; Tay et al., 2000). Recent studies have demonstrated that the induction of a temporally early class of Xenopus maternal mRNAs, including the Mos mRNA, is directed by PRE sequences in a CPE- and CPEB1-independent manner (Charlesworth et al., 2002; Charlesworth et al., 2004). By contrast, induction of CPE- and CPEB1-dependent mRNAs occurs temporally late during oocyte maturation (Charlesworth et al., 2002; Charlesworth et al., 2004). While consensus CPE sequences are present in the Mos mRNA 3′ UTRs from a variety of vertebrate species, it remains to be determined if PRE sequences also contribute to the temporal regulation of Mos mRNA translational activation in higher vertebrates.
Previous studies employing inhibition of protein synthesis in general or targeted ablation of the endogenous Mos mRNA, have suggested a role for regulated Mos mRNA translational control during human oocyte maturation (Hashiba et al., 2001; Pal et al., 1994). However, while a human CPEB1 protein has been characterized and shown to be expressed in human oocytes, the prior art is deficient in determining if regulated cytoplasmic polyadenylation of the maternal Mos mRNA occurs during human oocyte maturation. This study fulfills this long-standing need and desire in the art by showing that the human Mos 3′ UTR contains a functional CPE sequence, interacts with the human CPEB1 protein and directs maturation-dependent cytoplasmic polyadenylation of the endogenous Mos mRNA. Unlike the Xenopus Mos mRNA, there is no evidence for PRE-directed regulation of the human Mos mRNA. The results presented herein suggest fundamental differences in 3′ UTR regulatory element composition reflecting the differential temporal requirements for Mos mRNA translation during Xenopus and mammalian oocyte maturation.